1. Field of the Invention
The present invention relates to neocarzinostatin derivatives having excellent anticancer activity, which have the following formula EQU (SMA)-(NCS)-(SMA) (I)
wherein (NCS) represents divalent neocarzinostatin residue in which one hydrogen atom is removed from each of the primary amino group in alanine residue at the N-terminal of neocarzinostatin and that in lysine residue at 20th position from the N-terminal of neocarzinostatin and (SMA) represents monovalent styrene-maleic acid copolymeric residue having a weight-average molecular weight of 800.about.2,500 and consisting of structural units of styrene residue ##STR1## maleic acid residue ##STR2## and a residue having the following formula in which a hydroxyl group of one carboxyl group in the maleic acid residue is removed and linked to neocarzinostatin residue ##STR3## wherein the linkage of carbon atom in carbonyl group bonds to the neocarzinostatin residue, and a process for manufacturing the same.
2. Description of the Prior Arts
Neocarzinostatin (abbreviated as "NCS" hereinafter) is a proteinaceous anticancer agent produced in a medium in which Streptomyces carzinostaticus var. F-41 Kuroya is cultured (Japanese Patent Publication No. 42 (1967)-21,752 and U.S. Pat. No. 3,334,022). With respect to the primary structure of this substance, the total number of amino acid residues and estimated molecular weight have been reported to be 109 and 10,700 respectively, by Hiroshi Maeda who is one of the present inventors (Science, 178, 875-876 (1972) and Arch. Biochem. Biophys., 163, 379-385).
In the treatment of cancer, the metastasis of cancer cells is important and particularly the metastasis of said cells into lymphatic system is the most important problem. A wide variety of neocarzinostatin derivatives have been examined with the aim of lowering the toxicity, prolonging the biological activity of the drug in vivo, and directing its delivery more to the lymphatic system. As a result, it has been found that neocarzinostatin derivatives having the following formula (II), which is obtained by reacting two primary amino groups present in the molecule of neocarzinostatin with a partially hydrolyzed styrene-maleic anhydride copolymer having a molecular weight of 2,500.about.80,000 (the partially hydrolyzed styrene-maleic anhydride copolymer is abbreviated as "H-SMA" hereinafter), possess the above described activities and this has been disclosed in U.S. Pat. No. 4,182,752. EQU (SMA')-(NCS)-(SMA') (II)
wherein (NCS) means the same NCS residue as in the above described formula (I) and (SMA') means styrene maleic acid copolymeric residue having an average molecular weight of 2,500.about.80,000.
However, the above described NCS derivatives may contain various molecular species even after purification by the gel filtration method and the like. Concerning this point, the inventors have studied more rigorously and found that when NCS is reacted with H-SMA in an aqueous medium, the reaction solution after the completion of the reaction includes, other than the aimed NCS derivative, styrene-maleic acid copolymer produced by ring-opening hydrolysis of the maleic anhydride ring in H-SMA (referred to as "ring-opening hydrolyzed" product of H-SMA" hereinafter), unreacted NCS, an intermediate product to NCS derivative (I) having the following formula (III) EQU (NCS')-(SMA") (III)
wherein (NCS') represents monovalent NCS residue in which one hydrogen atom is removed from each of the primary amino group in alanine residue at the N-terminal of neocarzinostatin and that in lysine residue at 20th position from the N-terminal of neocarzinostatin, and (SMA") represents monovalent styrene-maleic acid copolymeric residue consisting of styrene residue, maleic acid residue and a residue having a formula in which hydroxyl group in carboxyl group has been replaced by nitrogen of the said amino group in NCS residue, and by-products having the following formulae (IV), (V) and (VI) ##STR4## wherein (NCS) has the same meaning as in the formula (I), (NCS') has the same meaning as in the formula (III), (SMA") has the same meaning as in the formula (III), and (SMA"') represents bivalent styrene-maleic acid copolymeric residue comprising styrene residue, maleic acid residue and two residues, each having a formula in which hydroxyl group is removed from one carboxyl group in maleic acid residue and bonded to NCS residue and m represents 1 or 2, and these impurities cannot be satisfactorily removed in the separation and purification step, such as gel filtration and the like and are admixed in the aimed NCS derivatives.